Part:BBa_K3183050

pTRKH3

This is a shuttle vector often which has been previously used in Lactobacilli and Lactococcus.¹ It has been shown to work in conjunction with constitutive promoters, P-erm (BBa_K3183000), P-ldh (BBa_K3183002), and P-slp (BBa_K3183001), when fused to mGFP5 (BBa_K3183011).²

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found in sequence at 7723
  Illegal suffix found in sequence at 1
  Illegal EcoRI site found at 5869
  Illegal XbaI site found at 1026
  Illegal XbaI site found at 7293
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 5869
  Illegal EcoRI site found at 7723
  Illegal NheI site found at 5318
  Illegal NheI site found at 6451
  Illegal NheI site found at 7592
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NotI site found at 9
  Illegal NotI site found at 7729
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 5869
  Illegal EcoRI site found at 7723
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 7723
  Illegal suffix found in sequence at 2
  Illegal EcoRI site found at 5869
  Illegal XbaI site found at 1026
  Illegal XbaI site found at 7293
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 7723
  Illegal EcoRI site found at 5869
  Illegal XbaI site found at 1026
  Illegal XbaI site found at 7293
  Illegal XbaI site found at 7738
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NgoMIV site found at 360
  Illegal NgoMIV site found at 520
  Illegal NgoMIV site found at 874
  Illegal AgeI site found at 6537
  Illegal AgeI site found at 6861
  
• 1000
  COMPATIBLE WITH RFC[1000]

Characterisation

The Oxford 2019 iGEM team used the pTRKH3 vector for all our work in Lactobacillus reuteri We were able to achieve transformation of this strain, demonstrating transformed cells through growth on MRS erythromycin plates and MRS erythromycin broth. Transformation was also confirmed using colony PCR and fluorescence microscopy. Links for the work done

Constitutive Reporter Genes: BBa_K3183100, BBa_K3183028, and BBa_K3183101
Constitutive CD27L and mClover3 Expression: BBa_K3183102
Constitutive AgrAC Expression: BBa_K3183103)

References

1. O'sullivan, Daniel J., and Todd R. Klaenhammer. “High- and Low-Copy-Number Lactococcus Shuttle Cloning Vectors with Features for Clone Screening.” Gene, vol. 137, no. 2, 1993, pp. 227–231., doi:10.1016/0378-1119(93)90011-q. 2. Lizier, Michela, et al. “Comparison of Expression Vectors in Lactobacillus Reuteri  Strains.” FEMS Microbiology Letters, vol. 308, no. 1, 2010, pp. 8–15., doi:10.1111/j.1574-6968.2010.01978.x.